Product Description
This kit is designed for the optimization of the cultivation process of fastidious microorganisms. Gut3Disks are presented here as additives to enhance the biomass yield and microbial viability facilitating the production of probiotics and live biotherapeutics. The protocol describes how to supplement a planktonic microbial culture with Gut3Disks and how to prepare the samples for downstream analyses. Downstream assays include cell viability assessment, flow cytometry, metabolomics, and sequencing. The potential applications of Gut3Gel include microbial mining and optimization of the production of probiotics and live biotherapeutics.
Product Page: https://www.bac3gel.com/category/gut3beads
Components
3x flasks, each containing 10 mL Gut3Disks.
50 mL Bac3Gel® dissolution medium (200 mM sodium citrate).
Material/Reagent Required But Not Provided
Sterile spatula with spoon.
0.9% (w/v) NaCl solution prepared in deionized water.
Any required culture medium.
Storage Conditions
Store in a dry place inside tightly sealed containers at 2-8 °C.
This product can be stored for at least 9 months.
Precautions and Disclaimer
For R&D use only.
Procedure
Before you begin
The protocol below describes the specific steps for enhancing growth and viability of single-strain planktonic bacterial cultures under laboratory-scale batch fermentation conditions. However, Gut3Disks have also been applied to controlled bioreactor systems and other dynamic culture environments. The Gut3Disks used in these protocols have dimensions between 4.5 to 6.0 mm. Gut3Disks’s composition is fully tunable; customized compositions are commercially available (i.e. incorporation of other mucin types, proteins, lipids and media).
Ensure all instruments and consumables (pipettes, falcons, spatula, and microcentrifuge tubes) are sterilized and free of contaminants.
All manipulations should be performed under aseptic conditions, ideally in a Class II biological safety cabinet.
Prepare a 0.9% (w/v) sterile NaCl solution.
A. Preparation of Microbial Suspensions
1. Grow cultures of the species of interest (e.g. Akkermansia muciniphila) to the logarithmic (log) growth phase in the appropriate medium.
Centrifuge the cultures to pellet the cells, then discard the supernatant.
Resuspend the cell pellet in an appropriate culture medium.
Measure the optical density at 600 nm (OD600) of the microbial suspension.
Adjust the cell concentration as needed.
B. Supplementing with Gut3Disks
The beads are supplied submerged in a 0.9% (w/v) NaCl solution. Keep this solution if you need to store the beads.
1. Dispense the Gut3Disks from the flask using the provided spoon.
Transferring the beads first into a Petri dish may help with controlling the dosage.
2. Supplement cell cultures with Gut3Disks at an amount equivalent to 10% of the total culture volume, based on their hydrated volume.
3. Incubate the samples at 37 °C for up to 72 hours under aerobic or anaerobic conditions, according to the experimental design.
C. Dissolution of Gut3Disks
1. Add Bac3Gel® dissolution medium (200 mM sodium citrate solution) in a 1:1 ratio to each sample. Mix thoroughly to fully dissolve Gut3Disks structure.
- The homogenized samples can be used for downstream assays.
For sequencing: Centrifuge the samples (14,000 x g, 5 minutes), then use the pellet for DNA extraction.
For metabolomics: Centrifuge the samples (14,000 x g, 10 minutes), then collect and lyophilize the supernatant.
For measuring metabolic activity or optical density: Centrifuge the samples (14,000 xg, 5 minutes), discard the supernatant, then resuspend in 0.9 (w/v) NaCl.
Other applications include assessing cell viability, performing flow cytometry analysis, and conducting standard microbiological characterization assays.